The thymus provides a unique microenvironment for the generation of self-tolerant, self-restricted T cells that are indispensable for the adaptive immune response. Given that thymus involution and reduced T cell output are consequences of advancing age or disease, there is considerable interest in therapeutic strategies aimed at thymus restoration. However, it is essential to understand the cellular interactions and molecular pathways that are required to generate the thymus de novo during ontogeny in order to devise rational approaches for its regeneration. This application focuses on the mechanisms by which neural crest-derived mesenchymal cells regulate development of the epithelial and hematopoietic compartments during thymus organogenesis. Our preliminary data reveal that neural crest cells (NCCs) play a previously unrecognized role in patterning 3rd PP endoderm and suggest that NCCs play distinct roles during early and late stages of thymus organogenesis. The NCC defect in Pax3Sp/Sp embryos results in a boundary shift between thymus and parathryoid specific domains of the common third pouch primordia. In comparison to wildtype littermates, the shared primordia of Pax3Sp/Sp mutants have a relatively smaller Gcm2 expressing domain and a correspondingly larger Foxnl expressing domain. Importantly, overall cellularity is comparable in the shared primordia of mutant and wildtype littermates. There is neither an increase in the frequency of proliferating cells, nor a decrease in the frequency of apoptotic cells in the shared primordia of Pax3Sp/Sp compared to Pax3+/+ littermates. Therefore, the increased number of third pouch endodermal progenitors that are committed to a thymus fate accounts for the unexpectedly large, albeit ectopic, thymic lobes in E12.5 Pax3Sp/Sp embryos. Although the findings appear to be at odds with the general consensus that NCC deficiencies result in thymus hypoplasia, the likely explanation for this paradox is that NCCs perform distinct functions at different developmental stages. Initially, NCC-derived signals are required for correct patterning of the third pouch endoderm. Specific Aims 1 and 2 focus on the molecular mechanisms by which NCCs control thymus versus parathyroid fate decisions in endodermal progenitors. At a slightly later stage of thymus organogenesis, the condensing NC-derived mesenchymal capsule produces Fgf7 and FgflO that promote-outgrowth of the thymus rudiment. It remains to be determined whether NCCs play additional roles in the late fetal and postnatal thymus. This issue is the central focus of Specific Aim 3.